The use of spore forming bacteria including certain Bacillus strains as probiotics for both humans and animals has become prevalent in recent years. As is noted in Knap et al. (WO 2010/070005) species such as Bacillus subtilis and Bacillus licheniformis are used as supplements in animal feed in order to promote growth by increasing the digestion and availability of nutrients from animal feed. Bacillus coagulans is the active ingredient in commercial probiotic products for human consumption, helping to aid in the digestion of proteins, lactose and fructose.
As is noted in Maathuis et al. (2010, Beneficial Microbes, 1(1): 31-36), such bacteria must be present in the small intestine in their germinated or vegetative form in order to function as probiotics. While such microbes are resistant to both stomach acid and bile salts in their spore form, they are susceptible to such environments in their vegetative states. Thus, if employed in their vegetative state, Bacillus strains must be contained within a pharmaceutically-acceptable acid-resistant or “enteric” carrier. See paragraph 7 of Farmer (US Patent Application 2003/0124104).
Unfortunately, it is difficult to formulate Bacillus species in their vegetative form such that they will possess an adequate shelf life. As is noted in GanedenBC product literature, traditional vegetative probiotics do not survive high heat and pressure in the manufacturing process, die quickly on the shelf, and are sensitive to stomach acids and bile enzymes in the gut. In contrast, formulations of such species in their spore form are much more suitable for commercial and practical use. Thus, as is noted by Cartman et al. (2008, Applied and Environmental Microbiology, August, p. 5254-5258) “[b]acterial spores are particularly well suited for use as live microbial products as they are metabolically dormant and highly resilient to environmental stress. These intrinsic properties are highly desirable from a commercial perspective and mean that spore-based products have a long shelf life and retain their viability during distribution and storage.”
The use of certain compounds, particularly certain L-amino acids, to stimulate the germination of Bacillus spores has been reported in the literature. Thus, for example, Foerster et al. (1966, Journal of Bacteriology 91(3): 1168-1177) discloses that the addition of L-alanine to spore suspensions in aqueous solutions will cause the germination of a number of Bacillus species. In addition, Maathius et al. cited above, suggests that the Bacillus coagulans spores in GanedenBC could be triggered into germination at the beginning of the small intestine by ingesting them together with a diet containing L-alanine, or by including L-alanine with such spores in a powder formulation. However, the approaches suggested by Maathius present several major challenges to establishing a probiotically effective bacterial culture:
1) Although the Bacillus coagulans spores employed in GanedenBC are themselves largely resistant to the low pH in the stomach, exposure to such acids could lead to a lag in germination when such spores enter into a more neutral pH. For example, Blocher et al. (1985, Applied and Environmental Microbiology 50(2): 274-279) demonstrated that B. cereus spores were inhibited from germinating at pH 4.5 even in the presence of the germinative compounds L-alanine or L-cysteine. Spores sequentially exposed to pH 4.5 buffer followed by pH 7.0 buffer were able to germinate upon exposure to such L-amino acids, but exhibited a lag in commitment to germinate. Any substantial delay in germination is highly undesirable, given the relatively short period of time that the spores may be present in the small intestines before being excreted. This is particularly true in smaller animals such as chicks, which have feed transit times of about 1.5 hours when 1 day old and transit time of less than 2 hours when 7 days old (see B. C. Watson et al. (2006, Poultry Science 85: 493-497), and shrimp, which have a transit time of less than 90 minutes (see Beseres et al., 2005, Journal of Shellfish Research 24(1):301-308). Thus there remains a need for accelerating and increasing germination of bacterial spores under conditions of exposure to low pH, such as those found in the stomach.
2) Diets high in L-alanine may also be high in D-alanine. As noted by Atluri et al. (2006, Journal of Bacteriology 188(1): 28-36), and Blocher et al. (cited above), D-alanine is a powerful inhibitor of Bacillus germination. In addition, there can be large amounts of other germination inhibitors (e.g., other D-amino acids, inorganic and organic acids, fatty acids, and bile salts) present in the small intestine which could compete with L-alanine if mixed in a powder form with Bacillus spores. Thus, a need exists to develop a method of improving spore germination in the presence of germination inhibitors that may compete with germinative compounds.
Accordingly, it is an object of this invention to provide a bacterial spore formulation which is capable of providing such benefits.